Ultra-rare constrained missense variants in the epilepsies: Shared and specific enrichment patterns in neuronal gene-sets

Background Burden analysis in epilepsy has shown an excess of deleterious ultra-rare variants (URVs) in few gene-sets, such as known epilepsy genes, constrained genes, ion channel or GABAA receptor genes. We set out to investigate the burden of URVs in a comprehensive range of gene-sets presumed to be implicated in epileptogenesis. Methods We investigated several constraint and conservation-based strategies to study whole exome sequencing data from European individuals with developmental and epileptic encephalopathies (DEE, n = 1,003), genetic generalized epilepsy (GGE, n = 3,064), and non-acquired focal epilepsy (NAFE, n = 3,522), collected by the Epi25 Collaborative, compared to 3,962 ancestry-matched controls. The burden of 12 non-synonymous URVs types in 92 gene-sets was compared between epilepsy cases (DDE, GGE, NAFE) and controls using logistic regression analysis. Results Burden analysis of brain-expressed genes revealed an excess of different URVs types in all three epilepsy categories which was largest for missense variants in highly constrained sites (constrained missense variants). The URVs burden was prominent in neuron-specific, synaptic and developmental genes as well as genes encoding ion channels and receptors, and it was generally higher for DEE and GGE compared to NAFE. The patterns of URVs burden in gene-sets expressed in inhibitory vs. excitatory neurons or receptors suggested a high burden in both groups in DEE but a differential involvement of inhibitory genes in GGE, while excitatory genes were predominantly affected in NAFE. Top ranking susceptibility genes from a recent genome-wide association study (GWAS) of generalized and focal epilepsies displayed a higher URVs burden in constrained coding regions in GGE and NAFE, respectively. Conclusions Using exome-based gene-set burden analysis, we demonstrate that missense URVs affecting mainly constrained sites are enriched in neuronal genes in both common and rare severe epilepsy syndromes. Our results indicate a differential impact of these URVs in genes expressed in inhibitory vs. excitatory neurons and receptors in generalized vs. focal epilepsies. The excess of URVs in top-ranking GWAS risk-genes suggests a convergence of rare deleterious and common risk-variants in the pathogenesis of generalized and focal epilepsies. ### Competing Interest Statement The authors have declared no competing interest. * ARC : Activity-Regulated Cytoskeleton protein. ATVB : Atherosclerosis, Thrombosis, and Vascular Biology Study. CAD : Coronary Artery Disease. CCR : Constrained Coding Regions. CDS : Coding sequences. dbGAP : Database of Genotypes and Phenotypes. DEE : Developmental and Epileptic Encephalopathies. FDR : False Discovery Rate. FMRP : Fragile-X Mental Retardation Protein. GATK : Genome Analysis Toolkit. GGE : Genetic Generalized Epilepsy. GO : Gene Ontology. GWAS : Genome-Wide Association Study. KEGG : Kyoto Encyclopedia of Genes and Genomes. ILAE : International League Against Epilepsy. MAC : Minor Allele Counts. MAF : Minor Allele Frequencies. MGI : Mouse Genome Informatics. MIGen : Myocardial Infarction Genetics Consortium. MPC : Missense Badness Polyphen and Constraint. MTR : Missense Tolerance Ratio. NAFE : Non-Acquired Focal Epilepsy. NDD : Neuro-Developmental Disorders. NMDA : N-methyl D-Aspartate. PCA : Principal Component Analysis. PCs : Principal Components. PPh2 : PolyPhen2 PSD-95 : Post-synaptic density protein 95. PTVs : Protein Truncating Variants. QVs : Qualifying Variants. SIFT : Sorting Intolerant From Tolerant URVs : Ultra-Rare Variants.